The invention relates to a method for early detection and diagnosis of cancer.
It is, today, an accepted dogma that a developing neoplasm is the result of genomic instability expressed by a multitude of changes in the genetic material (Loeb and Christians, 1996, Jackson and Loeb, 1998). The number of events required to occur for this process to culminate in a neoplasm has been estimated to be far in excess of that which can be accounted for by the normal mutation rate. It has therefore been suggested that carcinogenesis can occur only if the cancer-predisposed genome acquires a xe2x80x9cmutatorxe2x80x9d phenotype making it more mutable than its normal counterpart (Loeb 1991). Evidently, genomic instability depends on the fidelity of DNA replication, of DNA repair and of chromosome segregation. Indeed, consistent errors in DNA repair mechanisms resulting in multiple subtle changes at the nucleotide level were well documented in relation to oncogenesis. Similarly, a persistent damage in the segregating apparatus, causing DNA alterations at the chromosome level, expressed in an increased rate of losses and gains of whole chromosomes, was reported in connection with cancer (reviewed in Lengauer et al 1998a). Although, each of these errors enables the accumulation of multiple changes in the DNA compliment of an affected genome, these alterations are not too common. Changes at the nucleotide level appear only in a small portion of tumors, and the persistent damage caused to the segregating apparatus was, so far, observed in only few colon cancer cell lines (Lengauer et al 1998b).
An important aspect of DNA replication fidelity is the temporal control of the process. Accordingly, the specific time interval during DNA synthesis (S-phase of the cell cycle) at which a given DNA sequence is replicated appears to be a reliable indicator of transcriptional activity. However, it is not known yet whether the temporal order of replication is the cause or the effect of expression. Specifically, expressed DNA loci usually undergo early replication, while unexpressed ones tend to replicate late. This conclusion is based on several lines of evidence: (i) tissue-specific genes replicate early in cell types in which they are expressed and late in tissues in which they are silent (Selig et al. 1992, and reference therein); (ii) housekeeping genes, whose products are essential for cell maintenance, replicate early in most cells (Goldman et al. 1984; Holmquist 1987); (iii) DNA segments lacking transcriptional ability, such as satellite DNA, generally replicate late in S-phase (Selig et al. 1988; Ten Hagen et al. 1990, and references therein); (iv) the inactive X-chromosome in eutherian female cells is the last chromosome to replicate (Willard and Latt 1976, and references therein); and finally (v), most of the monoallelicaly expressed loci examined to date, manifest an allele-specific mode of replication, i.e., an early and a late replicating allele, in contrast to biallelically expressed loci which usually replicate highly synchronously (Ohlsson et al. 1998).
Most of the aforementioned cases were demonstrated by classical replication assays based on 5-bromodeoxyuridine (BrdU) incorporation, either in synchronized cells or in asynchronous cells fractionated by centrifugal elutriation, both followed by Southern blot hybridization of the newly synthesized BrdU-labeled DNA (reviewed in Boggs and Chinahult 1997). While it is possible to use these replication timing methods in the method of the invention, they are based on BrdU incorporation, and laborious, and require specific polymorphic markers or consistent differences in methylation levels for identification of individual alleles, which are not always available.
Using fluorescence in situ hybridization (FISH), it is possible to detect the presence of alleles and their replication status. A nonreplicated allele is detected as a single spot, whereas following replication, when the two chromatids are still located together, the allele is detected as a double spot. The presence of a single spot and a double spot in the same cell (hereinafter referred to as SD cell) therefore indicates that the two alleles are not replicating simultaneously (asynchronous replication; Selig et al. 1992; Boogs and Chinault, 1997).
Asynchrony of imprinted loci was clearly documented by FISH in the Prader-Willi syndrome locus; the paternal allele replicates earlier than the maternal allele which is usually silent (Kitzberg et al. 1993). On the other hand, FISH showed that in cells of individuals with uniparental disomy for the Prader-Willi syndrome locus the two alleles replicated highly synchronously, revealing loss of the asynchronous pattern of replication characterizing imprinted loci (Knoll et al. 1994; White et al. 1996).
Recently, the present inventors demonstrated by FISH that homologous regions at the TP53, CMYC, HER2, and D21S55 loci, each known to accommodate genes associated with various aspects of malignancy, replicate highly synchronously in different types of normal diploid cells, such as peripheral blood lymphocytes (Amiel et al. 1997, 1998a), bone-marrow cells (Amiel et al. 1998a), and amniotic fluid cells (Amiel et al.1998b, 1999a). On the other hand, these same loci, when present in lymphocytes and bone marrow cells of patients suffering from blood malignancies (CLL, CML and lymphoma), show loss of replication synchrony, (Amiel et al. 1997, 1998a). In light of the tight association between allele-specific replication and allele-specific expression it is reasonable to assume that all four aforementioned loci when present in lymphocytes and bone marrow cells of patients suffering from hematological cancers are subjected to some epigenetic mechanism leading to monoallelic expression. This assumption may be supported by the finding that changes in the mode of expression of an imprinted gene, a phenomenon often occurring in association with cancer and referred to as loss of imprinting (LOI), has been observed in lymphocytes and bone marrow cells of patients suffering from chronic myelogenous leukemia (Randhawa et al. 1998).
Moreover, the present inventors demonstrated by FISH that even the replication timing of homologous DNA counterparts lacking transcriptional ability is crucial for genomic stability. As, allelic conterparts of a-satellite DNA gaff (DNA associated with chromosomal segregation) replicate synchronously in cells showing an accurate segregation of chromosomes and asynchronously in cells displaying losses and gains of whole chromosomes (Litmanovich et al 1998).
It has now been surprisingly found that in peripheral blood lymphocytes of individuals stricken with solid tumors, genes and even non-coding DNA sequences changed the level of synchrony in replication timing of allelic counterparts. Allelic sequences replicating synchronously in cells of healthy subjects revealed a startling rise in asynchrony, while sequences replicating asynchronously in healthy subjects tended to replicate more synchronously in cells of cancer-stricken individuals. The change in timing of replication of non-coding loci was found to be associated with losses and gains of chromosomes (aneuploidy) a feature characteristic for cancer.
Furthermore, the exposure to various agents that interfere with gene expression and/or chromatin conformation further differentiate between lymphocytes of cancer patients and those obtained from non-cancerous subjects, as each usually alters the replication mode of only one type of cells, either cancerous or healthy, leaving the other un-touched.
It has been also demonstrated that synchrony in replication timing of allelic sequences may be used for the detection of causing genomic instability (genotoxicity) associated with cancer initiation, when applied in-vivo as well as in-vitro.
Thus, the phenomenon of modification in the inherent mode of allelic replication in the presence and in the absence of various DNA and chromatin modifiers may be useful in early diagnosis and detection of cancer.
Similarly, this phenomenon may be used for the detection of drugs and various environmental agents leading to genotoxicity.
In peripheral blood lymphocytes of individuals stricken with solid tumors, genes as well as noncoding DNA sequences changed the level of synchrony in replication timing of allelic counterparts. DNA sequences replicating synchronously in cells of healthy subjects revealed a startling rise in asynchrony, while sequences replicating asynchronously in healthy subjects tended to replicate more synchronously in cells of cancer-stricken individuals. It would therefore be useful to develop a method for using this phenomenon in early diagnosis and detection of cancer.
It would therefore also be useful to establish that loss of fidelity in the inherent temporal order of DNA replication provides a common source for generating numerous genetic events required for establishing a malignant phenotype.
The invention is directed at a method for the detection of cancer, appraising the prognosis thereof, and/or risk therefor comprising the steps of:
a) obtaining essentially non-malignant cells from an individual;
b) determining the coordination between ailelic counterparts of one or more loci in said cells. The cells are preferably subjected to a growth stimulus before step (b). Preferably, the cells are also subjected to drugs associated with gene expression and/or chromatin conformation before step (b). Also preferably, the cells are derived from a body tissue or body fluid. The body tissue is preferably bone marrow. The body fluid is preferably selected from blood, amniotic fluid, urine, and saliva. Preferably, the blood is peripheral blood. The cells are preferably lymphocytes.
The same method can be used for examine whether a drug and/or an environmental factor posses a genotoxic effect either applied in-vivo before step (a) or in-vitro before step (b).
The locus or loci are preferably expressed biallelically. Further preferably, the locus or loci are selected from tumor-associated genes. The tumor-associated genes are preferably selected from oncogenes, tumor suppressor genes, and transcription factors involved in translocations associated with blood tumors.
In another embodiment, the invention comprises a method as defined above wherein the locus or loci are expressed monoallelically. The monoallelically expressed locus or loci are preferably selected from imprinted loci, loci where one allele has been silenced, and loci on the X-chromosome in female individuals. The imprinted locus is preferably the Prader-Willi locus.
In another embodiment, the invention comprises a method as defined wherein the locus or loci are non-coding loci lacking transcriptional capability. The non-coding locus or loci are preferably selected from DNA sequences associated with chromosome segregation. The DNA is preferably satellite DNA.
In a more preferred embodiment of the invention, the locus or loci are selected from among HER2, CMYC, TP53, RB1, 21q22, GABRB3, SNRPN, D15S10, D22S75, DSTS WI-941, alpha, II and III satellites for all chromosomes.
The synchrony is preferably determined by fluorescence in situ hybridization.
The method of the invention is preferably a method wherein a change in synchrony indicative of cancer, the prognosis of cancer, or the risk therefor, is detected. The change in synchrony is preferably between about 3% and about 55%.
In one embodiment of the method of the invention, the change in synchrony is an increase in asynchrony. The increase is preferably between about 25% and about 30%. More preferably, the number of SD cells as determined by fluorescence in situ hybridization is increased by about 25% to about 30%.
In a further embodiment of the method of the invention, the change in synchrony is a decrease in asynchrony. The decrease is preferably about 15%.
In another embodiment of the method of the invention, synchrony is measured by fluorescence in situ hybridization, using a probe targeted to a biallelically expressed gene, in cells derived from peripheral blood, and wherein an increase in asynchrony of about 15% to about 35% is indicative of cancer, the prognosis thereof, or risk therefor.
In another embodiment of the method of the invention, synchrony is measured by fluorescence in situ hybridization, using a probe targeted to a monoallelically expressed gene, in cells derived from peripheral blood, and wherein a decrease in asynchrony of about 15% to about 25% is indicative of cancer, the prognosis thereof, or risk therefor.
Further, according to the present invention, there is provided a method for the detection of cancer and cancer risk by analyzing the replication status of a locus or loci within isolated cells whereby an altered replication status corresponds to cancer or cancer risk. Also provided is a method of determining the replication status of various DNA sequences by assaying isolated cells following in-vivo or in-vitro exposure to various drugs or environmental agents to determine genotxicity if any of the applied agents. A diagnostic test for detecting cancer or the risk of cancer having an allelic replication viewing device for viewing the mode of allelic replication of a DNA entity, a standardized table of replication patterns and an analyzer to determine an altered pattern of replication, whereby such altered pattern is a cancer characteristic is also provided. There is also provided a method for differentiating between hematological and solid malignancies by analysing the replication status of mono allelic expressed genes and analyzing the replication status of the sequences to distinguish between hematological and solid malignancies.